In vitro culturing of cells provides material necessary for research in pharmacology, physiology, and toxicology. The environmental conditions created for cultured cells should resemble as closely as possible the conditions experienced by the cells in vivo. One example of a suitable environment for culturing cells is a common laboratory flask such as demonstrated in U.S. Pat. No. 4,770,854 to Lyman. The cells attach to and grow on the bottom wall of the flask, immersed in a suitable sustaining media. The flask is kept in an incubator to maintain it at the proper temperature and atmosphere.
Gas exchange, particularly the utilization of oxygen by the cells, is a factor that limits the area for cell growth within a cell culture flask. Since flasks for cell culture typically grow attachment dependent cells in a monolayer roughly equal in size to the footprint of the flask, media volume is restricted to an area within the flask permissive to the diffusion of oxygen. Oxygen and carbon dioxide are of particular importance to the culturing of cells. The supply of oxygen for cellular respiration and metabolic function in conventional cell culture containers occupies the head space of the container, e.g., the void space in the container that is above the surface of the cell culture medium. Thus, the volume of the container and the surfaces within conventional cell culture containers are inefficiently used. This results in limiting the rate of gas exchange and/or restricting the equilibration of gases.
The need for large quantities of cells for high throughput screening (HTS) cell based assays continues to motivate organizations to search for methods to achieve larger cell numbers with minimal investment. The challenge is to generate large quantities of cells that all behave the same in cell based assays. In order to provide a solution it is imperative that the cells generated using such methods exhibit similar characteristics such as growth kinetics and response to stimuli. The multilayer flask that is the subject of commonly assigned patent application US 20070026516, incorporated in its entirety herein by reference, describes using several successive layers of cell growth chambers within a unitary vessel, each individual chamber having a gas permeable film and separated from the next by a tracheal space to provide gas exchange between the cells and culture medium and the atmospheric environment surrounding it. This allows for an expansive cell growth surface area when compared to standard traditional flasks such as the industry standard T175 flask. The HYPERFLASK (Corning Incorporated, Corning, N.Y.) vessel has roughly the same dimensions of the industry standard T175 flask (a flask body of approximately 157 mm×122 mm×53 mm) but was developed to generate approximately 10 times as many cells due to the 10 cell growth chambers housed within the flask body. A single HYPERFLASK vessel can be used to seed as many as 100 384 well microplates.
In a commercial embodiment of the HYPERFLASK product, each of the layers or individual cell growth chambers are held together by welds around the perimeter of the flask and press fit to each other in the center such as to form two press fit columns. Under certain conditions of use, pressure can build such that the press fit features cannot hold, and the columns separate leading to potential leaks in the vessel.